1. Field of the Invention
Pseudorabies is a fatal and economically important disease in swine. Because the incidence and severity have increased during recent years, a strict quarantine is required by law on all infected herds. Any movement of breeding pigs, except to slaughter, is required to be negative of infection by serological test. A simple, reliable and rapid testing method in the field, which can be applied by veterinarians without special facilities or skills, is needed.
This invention relates to a method and test kit which have such advantages and will contribute to the control of this worldwide disease in which early detection and elimination of the infected animals are important in the control. More particularly, this invention relates to a radial immunodiffusion enzyme assay technique and kit for the diagnosis of pseudorabies in swine by the detection of antibodies to pseudorabies virus. The technique is simple and inexpensive to perform and gives the results overnight. Because of its simplicity the test can be carried out on the farm by persons with little laboratory experience. Although pseudorabies affects other species, such as cattle, sheep and dogs, and the test disclosed can be used to diagnose the disease in them, the greatest present interest is in swine and the invention is described with particular reference to swine.
2. The Prior Art
Several techniques are available to test for pseudorabies virus antibodies. The virus neutralization test has been approved as the official method in the United States. However, for the virus neutralization test each sample must be sent to a laboratory which has facilities for tissue culture and techniques to conduct the test. The cost per sample is high. Obtaining the results requires 72 hours plus mailing time. Total time often exceeds 7 days. Other available tests include immunofluorescence, enzyme-linked immunosorbent assay (ELISA), agar gel immunodiffusion, complement fixation, indirect hemagglutination and skin tests. However, none of them is both easy to perform in the field and reliable. Details of each technique are described by Gustafson, Chapter 14, "Pseudorabies" in Diseases of Swine, edited by A. D. Leman et al, 1981, Iowa State University Press.
Although the basic principles of radial immunodiffusion enzyme assay are known and have been used for other purposes (Elwing et al, Journal of Immunological Methods, Vol. 39, pp. 247-56, 1980), the basic technique has been modified and adapted in order to permit testing of pseudorabies virus antibodies. Intact pseudorabies virus particles neither adhere to the test plate reproducibly nor react in the test with high reproducibility, sensitivity and specificity. The design has been improved by chemical treatment of infected cellular pseudorabies virus antigen. This is a significant step in eliminating the contamination of infectious virus in the environment and increasing the specificity of test reaction. Following infection, protective antibodies are directed against the envelope protein of pseudorabies virus, the antigen used in carrying out the present invention. Herein the viral component involved in protective immunity is specifically identified, rather than virus reactive or virus associated antigens. The manner of application of antigen to a test plate forming an integral part of the test kit has been modified to insure consistent binding of the antigen to the plate surface.